Project Summary/Abstract Recent publications indicate that post-transcriptional regulation makes an important contribution to circadian rhythms, in flies and in mammals. This evidence is now quite extensive and includes translational regulation, which impacts the synthesis of key clock proteins within the mammalian SCN as well as within the small number of central brain neurons that govern Drosophila circadian locomotor activity rhythms. These neurons pose a significant challenge ? and opportunity ? for circadian biology and for molecular biology more generally: how can one assess biochemical events within a few neurons? For almost all measurements, there is not enough signal and/or a problematic signal:noise ratio when an extract is made and assayed starting with only a few cells/brain. Even methods that tag brain neurons are plagued with this problem if the fraction of tagged neurons/brain is very low. To address this issue in the context of RNA binding proteins and their targets, my lab developed a new technique, which we call TRIBE (Targets of RNA Binding proteins Identified by Editing). It fuses the catalytic domain of the RNA editing enzyme ADAR to RNA binding proteins. TRIBE takes advantage of the fact that one can make clean RNA and sequence it even from a single cell despite the inability to work with an extract from the same source material. We have used TRIBE with three different RBPs and also showed that it can work within the ?small number of specific neurons? context. We have recently validated TRIBE and improved its efficacy, so that it has many fewer false negatives, i.e., it recognizes a much greater fraction of RBP- associated mRNAs. We will extend the method in two new translation-centric directions: to identify specific targets of the important translation factor eIF4E-BP and to identify ribosome-associated transcripts. We also propose to address some remaining issues, and extend TRIBE in new directions, with CRISPR-mediated knock-ins as well as with dimerization approaches. The latter will bring the editing moiety to the RNA on command, i.e., in response to a drug- or light-mediated dimerization signal. In all cases, assaying successfully small numbers of discrete neurons is the key biological focus. It is an important goal relevant to many human health problems like mental illness, neurodegeneration, stroke, substance abuse and addiction.